Regulatory T cells are the main cell population involved in the prevention or suppression of unwanted overt inflammatory processes. At the intestinal surface, which is chronically exposed to stimuli from both microbes and diet, peripheral Foxp3+ T cells (pTregs) are thought to prevent the development of inflammatory bowel diseases and food allergies. Surprisingly, very few Tregs are found in the most exposed layer of the intestinal mucosa: the single-cell layered epithelium. Yet, the understanding of immune regulatory processes at this layer is of utmost importance for a better comprehension of how pathological inflammatory processes arise, and for the development of novel intervention strategies. During the first four years of funding for this project we described environmental cues, transcription factors, and functional consequences related to the adaptation of CD4 T cells to the gut epithelium. We found that CD4 T cells belonging to different T helper lineages convert into CD8??+CD4+ intraepithelial lymphocytes (CD4-IELs) in a microbiota-, retinoic acid-, IFN-? and TGF-?- dependent manner. This unexpected T cell plasticity was also observed among total Tregs, which lose Foxp3 and convert to CD4-IELs upon migration to the epithelium, an effect attributed to the loss of the transcription factor ThPOK. Importantly, our work also suggested that pTregs and CD4-IELs perform complementary roles in the regulation of intestinal inflammation. Our preliminary repertoire analysis indicates strong TCR biases in both pTregs and CD4-IELs in the epithelium. However, the role of TCR stimulation for epithelial CD4 T cell residency and plasticity, the diversity of TCR repertoire and the nature of TCR ligands in epithelial CD4 T cell populations remain to be determined. We hypothesize that intra-epithelial adaptation of anti-inflammatory T cells is shaped by their TCR and by factors found in the epithelium, determining gut T cell functional specialization. Based on extensive published and unpublished data, this proposal undertakes several intertwined aspects of CD4 T cell adaptation to the gut epithelium, bringing together both conceptual and technological innovations. Using a combination of intersectional genetics, single-cell repertoire analyses and in vitro NFAT assays, we will define the TCR repertoire and ligands of epithelial CD4 T cells. Our unpublished data suggest a coordinated response involving multiple transcription factors that regulate Treg stability, tissue residency and CD4-IEL function. We will perform RNA-seq, ChIP-seq and ATAC-seq, combined with mouse genetics, to define the molecular machinery involved in the transcriptional changes observed in CD4 T cells that undergo epithelium-specific adaptation. Finally, we will define the role of continuous ThPOK and TCR expression in Treg suppressive function and whether CD4 T cell ?IEL differentiation? is important for immune regulation in the epithelium both at steady state and in models of intestinal inflammation. Hence, this proposal aims to characterize entirely novel immune regulatory pathways in the gut as well as to identify molecular mechanisms that determine plasticity of intestinal CD4 T cell lineages and their functional relevance.